Long chain polyprenyl phosphates have been described as carriers of carbohydrate units in the biosynthesis of cell wall polysaccharides in procaryotes and glycoproteins in eucaryotes. This research proposal is designed to extend our previous studies on the purification and characterization of Lactobacillus plantarum undecaprenyl pyrophosphate synthetase, including studies on possible membrane assoication and the mechanism of the formation of its unique product, which has a mixture of both trans and cis double bonds. The synthetase will be purified to homogeneity by a variety of procedures such as gel electrophoresis, isolelectric focusing and chromatography on hydrophobic column supports. The enzyme specificity with respect to stereochemistry and chain length of the allylic pyrosphosphate product will be investigated using a number of natural and synthetic allylic polyprenyl pyrophosphate substrates. Extensive studies will be made to characterize the phospholipid requirement with particular interest on the possibility that phospholipid fluidity as well as charge is important. The enzymic product will be isolated and purified by ion exchange, thin layer and gas chromatography. The product will be characterized with respect to both its chain length and stereochemistry by mass spectroscopy, double label isotope experiments and incorporation of label from delta 3-2R-H3 and delta 3-2S-(2H3)-isopentenyl pyrosphosphate. Two derivatives of isopentenyl pyrophosphate, isopentenyl imidodiphosphate and isopentenyl methylene diphosphonate will by synthesized and tested as substrates and inhibitors for the synthetase. Undecaprenyl pyrophosphate analogues will be tested as substrates and inhibitors for the synthetase. Undecaprenyl pyrophosphate analogues will be tested as substrates or inhibitors of an undecaprenyl pyrophosphate phosphatase isolated from M. lysodeikticus. The enzyme undecaprenol phosphokinase will be characterized from L. plantarum. A determination of any phospholipid requirement will be made comparing any similarities with the phopholipid requirements observed with the undecaprenyl pyrophosphate synthetase.